VAAM-Jahrestagung 2011 Karlsruhe, 3.–6. April 2011

equiring only magnesium as cofactor. Thus, we have identified a minimalset of factors essential for efficient membrane fusion.CBP008The MreB-like Mbl protein of S. coelicolor A3(2) requiresMreB for proper localization during spore wall synthesisA. Heichlinger*, A. Latus, W. Wohlleben, G. MuthDepartment of Microbiology/Biotechnology, Eberhard-Karls-University,Tübingen, GermanyThe majority of rod-shaped bacteria contain an actin-like cytoskeletonconsisting of MreB polymers which form helical spirals underneath thecytoplasmic membrane to direct peptidoglycan synthesis for elongation ofthe cell wall. In contrast, MreB of Streptomyces coelicolor is not requiredfor vegetative growth, but has a role in sporulation [1]. Beside MreB, S.coelicolor encodes two further MreB-homologous proteins, Mbl andSCO6166, whose function is unknown. Whereas MreB and Mbl are highlysimilar, SCO6166 is shorter, lacking subdomains IB and IIB of actin-likeproteins.We showed that MreB and Mbl are not functionally redundant but cooperatein spore wall synthesis. Expression analysis by semi-quantitative RT-PCRrevealed distinct expression patterns. mreB and mbl are predominantlyinduced during morphological differentiation, whereas sco6166 is stronglyexpressed during vegetative growth but switched off during sporulation.In contrast to rod shaped bacteria, deletion of mreB and/or mbl is tolerated inS. coelicolor. Vegetative growth was not affected but parts of the aerialhyphae lysed, spores were swollen and germinated prematurely. Themutants were also more sensitive to high salt concentrations. Whereas S.coelicolor M145 was still able to grow on LB supplemented with 6% NaCl,growth of ΔmreB or Δmbl mutants was abolished. Deletion of sco6166 hadno effect on morphological differentiation and its role in sporulation isunclear up to now.During aerial mycelium formation an Mbl-mCherry fusion proteincolocalized with an MreB-eGFP fusion protein at the sporulation septa.Whereas MreB-eGFP localized properly in the Δmbl mutant, Mbl-mCherrylocalization depended on the presence of a functional MreB protein.Our data suggest that Streptomyces requires mreB and mbl formorphological differentiation probably to build up a thickenedpeptidoglycan spore wall able to resist detrimental environmentalconditions.[1] Mazza, P. et al. Mol Microbiol. 2006. 60:838-852.CBP009Impact of membrane-perturbing antimicrobial peptideson bacteria visualized by electron microscopyM. Hartmann* 1 , M. Berditsch 1 , D. Gerthsen 2 , A.S. Ulrich 31 Institute for Organic Chemistry, Biochemistry, Karlsruhe Institute ofTechnology (KIT), Karlsruhe, Germany2 Laboratory for Electron Microscopy, Karlsruhe Institute of Technology(KIT), Karlsruhe, Germany3 Institute of Biological Interfaces (IBG-2), Karlsruhe Institute ofTechnology (KIT), Karlsruhe, GermanyThe effect of membrane-perturbing antimicrobial peptides (AMPs) has beenstudied extensively in the last decades, but the exact mode of action is yetnot fully understood. We therefore visualized the impact of tworepresentative cationic amphiphilic AMPs on bacteria using transmission(TEM) and scanning electron microscopy (REM). The peptide PGLa is α-helical and carries 5 positive charges, while Gramicidin S has a cyclic β-stranded structure with two cationic side chains. Their minimal inhibitionconcentrations (MIC values) were determined in salt-free medium for tworepresentative Gram-positive and Gram-negative bacterial strains, E. coliATCC 25922 and S. aureus ATCC 25923. For the EM samples, bacteriawere treated with sub- and supra-MIC concentrations, and fluorescencemicroscopy using SYTO9/propidium iodide confirmed that at supra-MICthe membrane integrity was disturbed, while at sub-MIC the cell membranesremained intact.After AMP treatment with either type of peptide, SEM revealed increasedturgidity of E. coli cells, and numerous bubbles and blisters formed on thecell surface. S. aureus cells were severely damaged, showing deep holes andburst cells. TEM revealed intracellular membranous structures in bothbacterial strains, probably as a result of lateral membrane expansion due topeptide insertion into the lipid bilayer. Additionally, the DNA region of S.aureus seemed to be compacted after AMP incubation.Treatment of E. coli in a medium with low ionic strength at sub- or supra-MIC led to highly turgid cells, compared to untreated controls. Thisobservation suggests that enhanced osmosis is facilitated across the innerbacterial membrane, before the more pronounced cell damages occur.Comparing our fluorescence and electron microscopy data, it is clear thatantimicrobial peptides render the bacterial membranes leaky even at sub-MIC concentrations, allowing small molecules like water to pass through,though not the larger propidium iodide. This means that even at lowconcentration the membrane permeabilizing effect of AMPs can result in areduced ability of the cells to regulate their osmotic pressure.[1] M. Hartmann et al (2010): Antimicrob. Agents Chemother. 54, 3132.CBP010Lipid Rafts in BacteriaD. LopezInstitute for Molecular Infection Biology, Infection Biology, Würzburg,GermanyQuestion: A feature common to all living cells is the presence of a lipidmembrane that defines the boundary between the inside and the outside ofthe cell. Proteins that localize to the membrane serve a number of essentialfunctions. In eukaryotic cells, membrane proteins that mediate signaltransduction and protein secretion are often localized in membranemicrodomains enriched in certain sterol lipids that are commonly referred toas „lipid rafts” (1, 2). Lipid rafts are required for the proper function of theharbored proteins. Thus, disruptions of lipid rafts are associated with a largevariety of human diseases including Alzheimer’s, Parkinson’s,cardiovascular and prion diseases (3). Up to now, lipid rafts have beenidentified and characterized in eukaryotic cells. However, many bacterialmembrane proteins involved in cell-cell signaling and signal transductionpathways are distributed heterogeneously across the cytoplasmic membrane(4), suggesting that specialized membrane microdomains are also a featureof bacterial cells.Results: Our work shows that bacteria contain lipid rafts functionallysimilar to those found in eukaryotes They harbor and organize proteinsinvolved in signal transduction, small molecule translocation and proteinsecretion. The lipids associated with the bacterial rafts are probablypolyisoprenoids synthesized via pathways that involve squalene synthasesbecause inhibitors of this enzyme interfere with the formation of lipid rafts.In addition, membrane microdomains from diverse bacteria harborhomologs of the protein Flotillin-1, a eukaryotic protein found exclusively inlipid rafts, responsible to orchestrate events occurring in lipid rafts. Amutant devoid of Flotillin-1 is defective in the signal transduction pathwayswhose sensor kinases are found in the rafts.Conclusions: Organization of physiological processes into microdomainsmay be a widespread feature in living organisms. On a more practical note,it is possible that lipid rafts can be exploited as a new target to controlbacterial infections because disrupting lipid rafts simultaneously affectsseveral key physiological processes associated with pathogenesis in differentbacteria.[1] D. Lingwood and K. Simons (2010): Science 327, 46.[2] Pike, L. J. (2006): J Lipid Res 47, 1597 (Jul, 2006).[3] Michel, V. and M. Bakovic (2007): Biol Cell 99, 129.[4] Meile, J. C. et al (2006): Proteomics 6, 2135.CBP011A role for the membrane curvature sensor DivIVA in cellseparation and virulence of Listeria monocytogenesS. Halbedel* 1 , B. Hahn 1 , R.A. Daniel 2 , A. Flieger 11 FG11 - Department of Bacterial Infections, Robert Koch Institute,Wernigerode, Germany2 Center for Bacterial Cell Biology, Newcastle University, Newcastle uponTyne, United KingdomDivIVA proteins are membrane binding proteins that are highly conservedamong the Firmicutes and the Actinomycetes. They have the remarkablefeature to accumulate at such areas where the membrane is most stronglybent and these are the invaginating septum at the site of cell division and thecell poles. Membrane binding is mediated via a unique dimeric lipid bindingdomain at the N-terminus that exposes two phenylalanine side chains to thesolvent which insert into the hydrophobic phase of the phospholipid bilayer.spektrum | Tagungsband 2011